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Background and objectives To examine anti-platelet autoantibodies in patients with immune thrombocytopenia (ITP) not only provides solid evidence for diagnosis, and also helps to select an individualized strategy for the treatment. The aim of this study is to develop a novel cell-based assay to detect autoantibodies in ITP patients. Methods/patients The DNA sequences of human platelet membrane protein GPIbα, GPIbβ, GP IX, GPIIb and GPIIIa subunits were obtained from NCBI database and synthesized. The synthetic fragments were ligated into pcDNA 3.3 and constructed the recombinant plasmids and transfected into Chinese hamster ovary (CHO) cells to establish cell lines stable expressing GPIb-IX and/or GPIIb/IIIa complexes. One hundred and two ITP patients with different anti-platelet autoantibodies, 57 patients with other kinds of autoimmune diseases and 104 healthy control were selected to examine sensitivity, specificity and accuracy of this method. Results CHO cells stable expressing GPIb-IX and/or GPIIb/IIIa proteins were established. The cells were fixed with 4% paraformaldehyde and stored at −80 ℃, more than 80% of the cells were still expressed target proteins after 180 days of storage. The concentrations of target antibody from 0.1 to 100 μg/ml were detectable by this method, and 10–50 μg/ml antibody binding to the CHO cells yielded higher distinguishable fluorescent intensities. Inter-assay and intra-assay coefficients of variation and receiver operating characteristic curve analysis showed that this method had relatively higher reproducibility and specificity. Compared with Flow Cytometric Immunobead Array, this method has relatively higher specificity (95.2%) and accuracy (90.8%) in detection of 102 ITP patients. Conclusion A novel cell-based assay to detect autoantibodies in ITP patients is established, which appears to be a promising method to diagnose ITP.